CN114585729A - Functional neuromodulation assembly - Google Patents

Abstract

Human midnuclear organoids or spheroids (hRNS) are produced in vitro and may be produced at least in part by human pluripotent stem (hPS) cells. Such spheroids mimic the human nucleus and contain specific groups of cells (e.g., serotonergic neurons) associated with the human nucleus, and can assemble with cortical spheroids (hCS) to form functional human neuromodulation circuits.

Description

Functional neuromodulation assembly

Cross-referencing

This patent application claims priority to U.S. provisional patent application No. 62/898,430, filed on 9/10/2019, the contents of which are incorporated herein in their entirety for all purposes.

Background

The mammalian neuromodulation system consists of discrete groups of neurons projecting from the brainstem, pons nucleus, or basal forebrain, which can fine-tune brain function and are associated with various neuropsychiatric disorders. Neuromodulatory agents such as serotonin, norepinephrine, acetylcholine, dopamine, and the like may act on these cells on multiple timescales, ranging from short-term modulation of neuronal and synaptic function to long-term circuit adaptation. During early brain development, neuromodulatory agents actively participate in the assembly of the nervous system by modulating cell division, differentiation, migration, synaptogenesis, synaptic transmission, and dendritic pruning. One of the earliest neuromodulatory innervation to the developing cerebral cortex was achieved by serotonergic neurons originating from the raphe nucleus located at the midline of the brainstem.

Although the serotonin (5HT) system in the human brain consists of only about 50 million neurons, it innervates almost every region of the central nervous system. Serotonin acts through at least fourteen different G protein-coupled receptors, and it has been demonstrated that these receptors in the human cerebral cortex differ from the corresponding receptors in other mammals and primates. Depending on the subtype, these receptors may exert inhibitory or excitatory effects on the regulation of neuronal activity.

Serotonergic neurons are produced in the human central nervous system as early as 5 weeks after conception (up to 15 weeks after conception), and the mesomeric nucleus already contains a stereotyped array of serotonergic neurons. Importantly, serotonergic neurotransmission dysfunction has long been associated with major depression (MDD), and many agents used in MDD, such as Selective Serotonin Reuptake Inhibitors (SSRIs), act by modulating this pathway. SSRIs are first-line drugs for the treatment of MDD. However, a significant proportion of patients remain resistant to SSRI, and it is not currently clear whether and how alterations in serotonergic neurons result in resistance to SSRI in these patients. In addition, serotonin signaling is associated with a variety of other neuropsychiatric disorders, such as schizophrenia, affective disorders, anxiety and autism spectrum disorders.

Region-specific brain organoids or brain spheroids do not currently contain a neuromodulation system, and there is currently no human platform to study, manipulate or explore neuromodulation pathways with human patient-derived cells.

Disclosure of Invention

The present invention provides compositions and methods for the in vitro production of human midnuclear organoids or spheroids (hRNS), which may be produced at least in part by human pluripotent stem (hPS) cells. Such spheroids mimic the human mid-suture and comprise specific sets of cells associated with the human mid-suture, e.g., serotonergic neurons, GABAergic neurons, etc.

The hRNS can functionally integrate with human cerebral cortical spheroids (hCS) comprising human cortical neurons (e.g., glutamatergic neurons) to provide cortical-raphocene-like assemblies (hCS-hRNS). The serotonergic neurons form bidirectional projections between the neurons of the hRNS and hCS to generate neuromodulation-like assemblies. The assembly-like bodies are composed of functionally integrated cells (including neurons) that interact in a physiologically relevant manner, e.g., forming synapses between classes of neurons, to provide physiologically relevant functional neural circuits. Using a combination of viral tracking and real-time imaging, the present invention provides evidence regarding the formation of human cortical-raphocene neural circuits generated in vitro, which provides useful modeling of cortical-raphocene pathway establishment and dysfunction.

In some embodiments, class assemblies are provided in which one or more of the cells can be genetically modified to provide additional screening functions. For example, one or both of cortical neurons and serotonergic neurons can be genetically modified to express a fluorescent calcium indicator, which is an indicator known and used in the art. One or both of cortical neurons and serotonergic neurons can be genetically modified to express light-activated opsin proteins. In some embodiments, the class of assemblies comprises serotonergic neurons expressing opsin proteins and cortical (e.g., glutaminergic) neurons expressing fluorescent calcium indicators, wherein the functional relationship between the neurons is evidenced by activating the serotonergic neurons using light and observing calcium indicator responses of the cortical neurons. There are 5HT lineage specific viral tools, such as AAV-based FEV mini-promoter driven reporter genes, and it has been demonstrated that the viral tools can be used to specifically probe and study 5HT lineage cells in hnns and hCS-hnns as follows.

The hnns spheroids and hCS-hnns like assemblies provide unique opportunities for analysis of: development and function of serotonergic neural circuits between the raphe nucleus and the cortex (or vice versa); and serotonergic regulation of cortical neural circuits. In addition, these spheroid or brain region specific organoids and organoid assemblies provide models to study the effects of neurological or psychiatric disorders on neural circuits in these brain regions. Of particular relevance are neurological or psychiatric disorders associated with serotonin dysfunction, such as MDD, schizophrenia and other psychoses, affective disorders (e.g., depression, bipolar disorder or anxiety) and Autism Spectrum Disorder (ASD).

In addition, these spheroids and class assemblies can be used to establish SSRI functional screening platforms, e.g., to mimic different pharmacological modulation of the serotonergic system by SSRI and atypical antipsychotics; analyzing serotonergic related diseases such as serotonin syndrome and the like; analyzing the effect of intra-uterine SSRI on cortical development; and so on. For example, in neuromodulation-like assembly systems in which cortical neuronal activity is monitored with calcium indicators (gCamp6 or Fluo-4) or voltage indicators and/or serotonergic neuronal activity is modulated with electrodes, optogenetics, or the like, the systems can be used to perform high-throughput assays on libraries of candidate agents, such as 5-HT receptor (e.g., antipsychotic drug) modulators or 5-HT transporter (SSRI) modulators, to test their relative physiological effects (i.e., calcium amplitude, calcium spike frequency, neuronal membrane voltage changes, etc.) as compared to drugs of known activity. The model provided by the present invention also allows testing the impact of genetic background, such as by using multiple assembly-like bodies from different human participants, optionally containing gene variants that affect the function of the receptor or transporter of interest. The composition of the 5HT receptors expressed by the postsynaptic neurons varies with the neuronal subtype and developmental age, determining the effect of stimulation and pharmacological application in this system, leading to alterations in the neuromodulatory response, such as inhibition or prolongation of excitation, rigidity, adaptation, cluster firing patterns. Comparison with one or more known control agents can be used to determine the desired response.

In addition, this system also has the advantage of providing the opportunity to use patient-derived hiPS cells. This may enable screening methods to elucidate the mechanism of SSRI resistance in hRNS-hCS-like assemblies derived from patients suffering from neurological or psychiatric disorders such as Major Depressive Disorder (MDD), drug resistance or intolerance to SSRI. In addition, control and patient cells (e.g., control-hCS versus patient-hnns) can be combined to generate assembly-like bodies to dissect cell autonomous contributions. This platform can also be used to study the genetic forms of autism spectrum disorders associated with deregulation of the 5-HT system, such as 16p11.2 microdeletions or duplications, and rare, causative mutations in the FEV gene.

The invention also provides a method for determining the activity of a candidate agent in the assembly-like in vivo neural circuit, the method comprising contacting the candidate agent with the assembly-like. The cells present in the assembly-like body optionally comprise at least one allele that encodes a mutation associated or likely to be associated with a neurological or psychiatric disorder, and determining the effect of the agent on morphological, genetic or functional parameters including, but not limited to, neuronal number, neuronal function, gene expression profile, cell death, single cell gene expression (RNA-seq), calcium imaging with pharmacological screening function, patch clamp recordings, regulation of synaptogenesis, and the like.

These and other objects, advantages and features of the present invention will be apparent to those skilled in the art upon a reading of the detailed information (see below) of the subject methods and compositions.

Drawings

The invention will be best understood from the following detailed description when read in connection with the accompanying drawings. It is emphasized that, according to common practice, the various features of the drawings are not to scale. On the contrary, the dimensions of the various features are arbitrarily expanded or reduced for clarity. Included in the drawings are the following figures:

FIGS. 1A-1G, (A) schematic diagrams showing hRNS-hCS assemblies (B) schematic diagrams showing formulations for deriving human mid-stitched nucleus-like spheroids (hRNS). (C) RT-qPCR analysis of genes expressed in human cortical spheroids (hCS) and developing hindbrain within hRNS. Different hipscs are represented by different colors. (D) Representative images of Immunocytochemistry (ICC) of the metacerebral progenitors and (E) cells of the 5-HT neural lineage are shown. (F) 5-HT neurotransmitter levels in hRNS were measured by HPLC under different in vitro phase and hipSC line conditions. (G) RT-qPCR analysis of different 5-HT receptors (HTR) in hCS at day 100 after in vitro differentiation. HTR subtypes are indicated by color.

FIGS. 2A-2C (A) UMAP projection of single cell transcriptome data from 13,708 hRNS cells harvested from 9 spheroids derived from 3 hipSC lines from day 79 to day 82 after in vitro differentiation. (B) The cluster marker expression for different neuronal populations in hRNS is shown. (C) FEV + sub-cluster analysis of individual sub-clusters with tail and beak characteristics (upper panel) and heatmaps of the top 10 differentially expressed genes in each cluster (lower panel) are presented.

FIGS. 3A-3C (A) assemblies of hCS and AAV-Syn1: mCherry-tagged hRNS (left panel). 3D reconstitution of hCS-hRNS-like assemblies with hRNS cells labeled with mCherry (right panel). (B) Shows the labeled NKX6-1 in hRNS-hCS assembly⁺Metabrain progenitor cells and TPH2⁺Representative ICC images of 5-HT expressing cells. (C) AAV-Syn1: assembly of mCherry-labeled hCS and AAV-Syn1: eYFP-labeled hRNS (left panel). Real-time imaging of bi-directionally projected doubly infected hRNS-hCS is shown (right panel). Inset shows the appearance of 5-HT common to forebrain-projections⁺Axonal morphology of spindle-like varicose veins in neuronal processes.

FIGS. 4A-4℃ (A) shows a schematic representation (left panel) of the characterization of 5HT lineage specific FEV reporter Ple67 on dissociated hRNS cells, representingSexual immunocytochemistry images (center panel) and colocalization quantification (right panel). (B) Assembly of mCherry-labeled hCS with AAV-Ple67iCRE and AAV-EF1 α -DIO-eYFP with AAV-SYN1: (left panel). Real-time imaging of infected hRNS-hCS projected towards 5HT lineage cells of hCS is shown (right panel). (C) Assembly of GCaMP7 s-labeled hRNS with AAV-Ple iCRE and AAV-EF1 alpha-DIO-ChRmine-Kv2.1-mScalet labeled hRNS (left panel) and stimulation of 5-HT lineage FEV in hRNS by 625nm light in hRNS neurons⁺Example of light induced calcium transient traces by cells (right panel).

The invention will be best understood from the following detailed description when read in connection with the accompanying drawings.

Detailed Description

Before the present compositions and methods are described, it is to be understood that this invention is not limited to the particular compositions and methods described, as such may, of course, vary from practice. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the inventive concept, the scope of which will be limited only by the appended claims.

Where a range of values is provided, it is understood that each intervening value, to the extent that there is a lower limit, is explicitly disclosed. Unless the context clearly dictates otherwise, each intermediate value should be as low as one tenth of the unit of the lower limit. The invention extends to each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and the invention also covers each range where one, none, or both limits are included in the smaller ranges, subject to the requirement of any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, some potential and preferred methods and materials are described below. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. It should be understood that, in case of conflict, the present disclosure should replace any disclosure in the cited publications.

It must be noted that, as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a reprogramming factor polypeptide" includes a plurality of such polypeptides, and reference to "the induced pluripotent stem cell" includes one or more induced pluripotent stem cells and equivalents thereof known to those skilled in the art, and so forth.

The publications discussed herein are provided solely for their disclosure prior to the filing date of the present patent. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

Definition of

By "pluripotent" and multipotent stem cells is meant that such cells are capable of differentiating into all types of cells within an organism. The term "induced pluripotent stem cell" encompasses pluripotent cells (e.g., Embryonic Stem (ES) cells) that can be cultured for a long period of time while maintaining the ability to differentiate into all types of cells within an organism, and also encompasses pluripotent cells (other than ES cells) derived from differentiated somatic cells, i.e., cells that have a narrower potential, are more defined, and cannot produce all types of cells within an organism without experimental manipulation. hiPS cells have a human ES-like morphology, grow as flat colonies, have a large nuclear to cytoplasmic ratio, well-defined borders and distinct nuclei. Furthermore, hiPS cells express several pluripotency markers known to those of ordinary skill in the art, including but not limited to alkaline phosphatase, SSEA3, SSEA4, Sox2, Oct3/4, Nanog, TRA160, TRA181, TDGF 1, Dnmt3b, FoxD3, GDF3, Cyp26a1, TERT, and zfp 42. In addition, the hiPS cells are capable of forming teratomas. Furthermore, they are capable of forming or contributing to ectodermal, mesodermal or endodermal tissues in living organisms.

As used herein, "reprogramming factors" refer to one or more, i.e., a mixture, of biologically active factors that act on a cell to alter transcription, thereby reprogramming the cell into a pluripotent or multipotent cell. The reprogramming factors can be provided to the cells, e.g., cells from individuals with a family history of heart disease or a genetic makeup of interest, such as fibroblasts, adipocytes, etc., alone or in a single composition of reprogramming factors (i.e., a pre-mixed composition). The factors may be provided in the same molar ratio or in different molar ratios. The factor may be provided one or more times during the culturing of the cells of the invention. In some embodiments, the reprogramming factors are transcription factors, including but not limited to Oct 3/4; sox 2; klf 4; c-Myc; nanog; and Lin-28.

The somatic cells are contacted with reprogramming factors as defined above, in a combination and amount sufficient to reprogram the cells into pluripotent cells. The reprogramming factors may be provided to the somatic cells alone or in a single composition of reprogramming factors (i.e., a pre-mixed composition). In some embodiments, the reprogramming factors are provided as multiple coding sequences on a vector. The somatic cells may be fibroblasts, adipocytes, stromal cells, and the like, as is well known in the art. Somatic or hiPS cells can be obtained from a cell bank, a normal donor, an individual with a neurological or psychiatric disease of interest, and the like.

After pluripotency induction, the hiPS cells are cultured according to any suitable method, such as on irradiated feeder cells and commercially available media. The hiPS cells can be dissociated from the feeder layer by digestion with a protease (e.g., dispase), preferably at a concentration and for a time sufficient to isolate intact pluripotent stem cell colonies from the feeder layer. The spheroids can also be generated from hiPS cells grown in feeder-free conditions by dissociating into single cell suspensions and aggregates using various methods, including centrifugation in plates, etc.

Genes can be introduced into the somatic cells or the hiPS cells derived therefrom for various purposes, e.g., replacing genes having loss-of-function mutations, providing marker genes, etc. Alternatively, a vector expressing antisense mRNA, siRNA, ribozyme, or the like is introduced, thereby blocking the expression of an unintended gene. Other gene therapy approaches have introduced drug resistance genes that favor normal progenitor cells and are subject to selective pressure, such as multiple drug resistance genes (MDR) or anti-apoptotic genes, such as BCL-2. As described above, various techniques known in the art can be used to introduce nucleic acids into the target cells, e.g., electroporation, calcium-precipitated DNA, fusion, transfection, lipofection, infection, and the like. The particular manner in which the DNA is introduced is not critical to the practice of the invention.

Disease-associated or disease-causing genotypes can be generated in healthy hiPS cells by targeted gene manipulation (CRISPR/Cas9, etc.), or hiPS cells can be derived from individual patients carrying disease-associated genotypes or diagnosed with disease. In addition, neurological and neuromuscular diseases with less defined or no genetic components can be studied in the model system. A unique advantage of this approach is that the edited hiPS cell lines have the same genetic background as their corresponding unedited hiPS cell lines. This reduces variability associated with inter-lineage differences in genetic background. The conditions of neurodevelopmental and neuropsychiatric disorders and neurological diseases with strong genetic makeup or directly caused by genetic or genomic alterations can be modeled with the system of the present invention.

The methods and compositions described herein are related to brain region-specific spheroids. Brain region-specific spheroids are three-dimensional (3D) aggregates of cells that resemble specific regions of the human brain and contain functional neurons commonly associated with this brain region. These spheroids can remain in suspension culture for extended periods of time without adhering to a surface (e.g., a petri dish surface), e.g., 2 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, or longer. By functional neuron is meant a neuron capable of forming a functional synapse with other neurons in the same spheroid or in another spheroid. Calcium imaging can be used to reveal the formation of functional synapses, as shown in more detail in the examples. For example, the human metakaryotic spheroids described herein comprise metakaryotic neurons, such as serotonergic neurons.

The methods and compositions described herein are also related to class assemblies comprising more than one (e.g., two or three or more) of these brain region-specific spheroids. The class assemblies described herein are similar to regions of the brain and contain functional neural circuits between neurons of one spheroid (representing one region) and neurons of another spheroid (representing another region). For example, the cortical-mesomeric assembly is similar to the cortex and mesomeric nucleus of the human brain and contains neurons (e.g., serotonergic neurons) projecting between the mesomeric spheroid and cortical spheroids, wherein these neurons are capable of forming functional synapses with human cortical neurons (e.g., glutamatergic neurons) of the cortical spheroids and modulating the activity of neural circuits in the cortical spheroids. Like the spheroids, these types of assemblies also hold for long periods of time without adhering to the surface.

And (5) a central suture core. The human raphe nucleus is a cluster of neurons located within the brain stem. Most of the neurons originating in the raphe nucleus are serotonergic neurons that project to multiple locations of the human central nervous system, including the cortex, ventral striatum, hippocampus, and amygdala of the forebrain. The raphe nucleus also receives junction information from the cerebral cortex and other brain regions. Through interaction with these areas and other neuromodulation systems, serotonin can affect a wide range of functions, such as reward assessment, impulse, nociception, and anxiety states. Impairment of these systems is associated with a variety of neuropsychiatric disorders, some of which are described in more detail below.

Serotonergic neurons are neurons that produce the neurotransmitter serotonin, also known as 5-hydroxytryptamine or 5-HT. The presence of these neurons can be detected by expression of, for example, serotonin, enzymes involved in the serotonin pathway (e.g., tryptophan 5-hydroxylase 2(TPH2)), and/or markers of mature serotonin neurons (e.g., vesicular monoamine transporter 2(VMAT2) and serotonin reuptake transporter (SERT)). Serotonin acts through at least fourteen different G protein-coupled receptors which, depending on their subtype, can exert excitatory or inhibitory neuronal activity.

The present invention provides in vitro spheroid structures (also known as region-specific organoids) comprising serotonergic neurons and class assemblies derived therefrom. The presence of serotonergic neurons can be verified by determining the presence of neurons expressing the above markers and the presence of serotonin produced by these neurons. The hRNS can comprise at least 1% (as a percentage of the total cell population) of serotonergic neurons defined by these markers, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, or more serotonergic neurons.

When the hnns is fused to hCS to generate assemblies, the constructs provide a model for serotonergic (5-HT) regulation of the cortical circuit. The functional integration of serotonergic and cortical neurons can be verified by the presence of bidirectional axonal projections by which axonal projections of hCS-derived neurons project to hnss and axonal projections of hnns-derived neurons project to hCS. Bidirectional axonal projection in hnns-hCS can be visualized by: hRNS and hCS were labeled with neuron-specific viral reporter genes (e.g., AAV-DJ-hSyn1:: mChrry for hCS and AAV-DJ-hSyn1:: eYFP for hRNS) prior to assembly and the appearance of projections monitored by long-term confocal imaging. A subset of these projections is expected to establish synaptic connections. Notably, in addition to synaptic connections, some serotonergic neurons release 5-HT in a diffuse manner without a tightly fitting postsynaptic site, such that cells distal to the release site can bind to the released 5-HT (referred to as "volume transfer"). Thus, a true measurement of serotonergic connectivity of hCS may also involve non-connective neurotransmitter transmission.

Functional assays for circuit integration may include, for example, determining signaling between neuron classes; and may include determining the effect of the neuromodulation system on regulation of cell division, differentiation, migration, synaptogenesis, and dendritic pruning. For example, in a optogenetic system, photostimulation of serotonergic neurons can reveal patterns of response in functionally integrated cortical neurons, e.g., increased calcium activity in response to photostimulation, decreased calcium activity in response to photostimulation transiently, or throughout the post-stimulation period; and so on. There are a number of reactions that may indicate functional connectivity and SSRI reactivity in hRNS-hCS (as described above). For example, the calcium response following stimulation can be determined, where the number of activated neurons per assembly can be 10 or more, 100 or more, 103 or more. A two-photon (2P) system with optimized imaging means can be used to capture a greater number of events.

The cerebral cortex. The adult cerebral cortex contains two major classes of neurons: glutamatergic cortical neurons (also known as pyramidal cells) and gabaergic interneurons.

Glutamatergic neurons. The mature cerebral cortex contains heterogeneous glutamatergic neuronal populations, which can constitute a highly complex histological architecture. So-called excitatory neurons are generally classified according to the layer in which their cell bodies are located, specific combinations of gene expression, dendritic morphology, electrophysiological properties, and the like. Gabaergic interneurons are inhibitory neurons of the nervous system, which play a crucial role in neural circuits and activity. They are known for their release of the neurotransmitter gamma-aminobutyric acid (GABA). Interneurons are a special type of neuron whose main role is to modulate the activity of other neurons in the neural network. Cortical interneurons are so named for their localization in the cerebral cortex.

The relevance of the disease. Serotonergic neural circuit dysfunction is associated with a variety of neurological and psychiatric disorders, including schizophrenia, affective disorders, and Autism Spectrum Disorders (ASD). The system described herein provides unique opportunities to study the role of these circuits in these disorders and allows screening for potential therapies.

Affective disorders are a group of mental disorders, also known as mood disorders, which include depression, bipolar disorder and anxiety disorders. Alterations in serotonin activity are associated with various mood disorders, and Selective Serotonin Reuptake Inhibitors (SSRIs) are frequently used as treatment regimens for affective disorders such as Major Depressive Disorder (MDD). The potential role of serotonin in affective disorders is not fully understood, and thus the system described herein provides the opportunity to further study its role in these disorders, screen for potential new SSRIs, and mimic the interaction between SSRIs and serotonergic neurons. Very high levels of serotonin can cause a disease known as serotonin syndrome, with toxic and potentially fatal effects, and can be further studied using the systems described herein.

Schizophrenia. Schizophrenia is a chronic, serious mental disorder that affects the behavior of an individual. The underlying cause of schizophrenia is still unclear, but the disorder is associated with abnormalities in serotonin and dopamine signaling in the central nervous system. The system described herein provides the opportunity to further investigate the role of serotonin in schizophrenia as well as to develop potential treatments.

Autism Spectrum Disorder (ASD) is a developmental disorder that affects and correlates with communication and behavior. Although the contribution of the serotonin system to the pathophysiology of ASD is not completely understood, elevated whole serum serotonin is the first biomarker found in ASD and is present in more than 25% of affected children (Muller et al, "serotonin system in autism spectrum disorders: from biomarker to animal model", neuroscience, 321: 24-41 (2016)). The system described herein provides the opportunity to study the role of serotonin in ASD.

A calcium sensor. Neural activity causes rapid changes in intracellular free calcium, which can be used to track the activity of neuronal populations. Art-recognized sensors for this purpose include fluorescent proteins that fluoresce in the presence of changes in calcium concentration. These proteins can be introduced into cells (e.g., hiPS cells) by including the coding sequences on a suitable expression vector (e.g., a viral vector) to genetically modify neurons produced by the methods described herein. GCaMP is a widely used protein calcium sensor consisting of fluorescent proteins such as GFP, calcium binding protein calmodulin (CaM), and M13 peptide that interacts with CaM, although a variety of other sensors are available. There are many different proteins available for use, including, for example, those described in the following publications: zhao et al, (2011) science, 333: 1888-1891; mank et al, (2008) nature: method, 5 (9): 805-11; akerboom et al, (2012) J Neuroscience, 32 (40): 13819-40; chen et al, (2013) nature, 499 (7458): 295-300; and the like; U.S. patent nos. 8,629,256, 9,518,980, 9,488,642 and 9,945,844.

Optogenetics integrates optical and genetic engineering to measure and manipulate neurons. The actuator is a gene coding means for light activated protein control; such as opsins and optical switches. Opsins are light-gated ion channels or pumps that absorb light at specific wavelengths. Opsins can be targeted to and expressed in specific subsets of neurons, allowing precise spatiotemporal control of these neurons by turning the light source on and off. Channelrhodopsin typically causes neurons to depolarize rapidly when exposed to light by directly stimulating ion channels. Chlamydomonas reinhardtii channel rhodopsin-1 (ChR1) is excited by blue light and when stimulated allows non-specific cations to flow into the cell. Examples of chrs from other species include: CsChR (from Chloromonas subdivisa), CoChR (from Chloromonas oogama) and SsChR (from Scherffelia dubia). Synthetic variants have now been created, such as ChR2(H134R), C1V1(t/t), schief; CheTA, VChR1, Chrimson, Chronos, PsChR2, CoChR, CsChR, CheRiff, and the like. Alternatively, ChR variants have been created and found that inhibit neurons, such as GtACR1 and GtACR2 (from the cryptophyte cyanobacterium) and variants such as iChloC, SwiChRca, Phobos, Aurora. Halophilic rhodopsin (called NpHR, from halophilic halophil monad) when triggered with yellow light hyperpolarizes the cell, and variants include Halo, eNpHR2.0, eNpHR3.0, Jaws. Archaerhodopsin-3 (Arch) from rhodobacter naxatilis is also used for neuronal inhibition.

The terms "treat," "treating," and the like, as used herein generally refer to obtaining a desired pharmacological and/or physiological effect. Prophylactic action means complete or partial prevention of a disease or a symptom thereof, and therapeutic action means partial or complete stabilization or cure of a disease and/or adverse reactions caused by a disease. As used herein, "treatment" encompasses any treatment of a disease in a mammal, particularly a human, including: (a) preventing a subject from developing a disease or developing symptoms, wherein the subject may be predisposed to developing the disease or developing the symptoms but has not yet been diagnosed; (b) inhibiting the disease symptoms, i.e., arresting their development; or (c) alleviating the symptoms of the disease, i.e., causing regression of the disease or symptoms.

The terms "individual", "subject", "host" and "patient" are used interchangeably herein to refer to any mammalian subject, particularly a human, in need of diagnostic, therapeutic or therapeutic treatment.

Method for producing spheroids and assembly-like bodies

The present invention provides methods for obtaining and using in vitro cell cultures of spheroids (also known as brain region-specific organoids) and assembloids produced by human pluripotent stem cells. Human midkine spheroids (hnns) were generated using a multi-step procedure. Various differentiated spheroid structures (e.g., hnns and hCS) are differentiated from neural progenitor spheroids. In some embodiments, the human pluripotent stem cell is an induced human pluripotent stem (hiPS) cell. In some embodiments, the hiPS cells are derived from somatic cells obtained from an unaffected individual. In other embodiments, the hiPS cells are derived from somatic cells obtained from an individual that comprise at least one allele encoding a mutation associated with a disease, including but not limited to the neurological or psychiatric disorders described above.

Human neural progenitor cell spheroids. The neural progenitor spheroids can be differentiated from pluripotent stem cells, including but not limited to human induced pluripotent stem cells, hiPS cells. Initially, the hiPS cells may be obtained from any suitable source, or may be generated from somatic cells using art-recognized methods. The hiPS cells are dissociated from the feeder layer, into individual cells and grown in suspension culture, preferably upon dissociation into intact colonies. In certain embodiments, the culture is a feeder-free culture, such as when grown on vitronectin-coated containers. The culture may further be free of non-human components, i.e., free of heterologous animal components. The hiPS cells may be cultured in any medium suitable for growth and expansion of hiPS cells. For example, the medium may be Essential 8 medium. Suspension growth optionally includes adding to the medium an effective dose of a selective Rho-associated kinase (ROCK) inhibitor for an initial phase of culture for up to about 6 hours, about 12 hours, about 18 hours, about 24 hours, about 36 hours, about 48 hours (see, e.g., Watanabe et al, (2007) Nature: Biotechnology, 25: 681686). Inhibitors useful for such purposes include, but are not limited to, Y-27632; thiazovivin (cell research, 2013, 23 (10): 1187-200), Fasudil (HA-1077) HCl (J.Clin. Res., 2014, 124 (9): 3757-66), GSK429286A (J.Sci.USA, 2014, 111 (12): E1140-8), RKI-1447, AT13148, etc. in particular embodiments, the ROCK inhibitor Y-27632 is used.

The suspension cultured hiPS cells were then induced towards neural fates. The culture may be a feeder layer-free culture. For neural induction, an effective dose of a BMP inhibitor and a TGF β pathway inhibitor is added to the medium (e.g., Essential 8 medium) for at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, and up to about 10 days, up to about 9 days, up to about 8 days, up to about 7 days, up to about 6 days, up to about 5 days. Such inhibitors are also known as SMAD pathway inhibitors. For example, an effective dose of Doxorphine (DM) that inhibits the Bone Morphogenetic Protein (BMP) type I receptors (ALK2, ALK3, and ALK6) can be added at a concentration of at least about 0.1 μ M, at least about 1 μ M, at least about 5 μ M, at least about 10 μ M, at least about 50 μ M, and at most about 100 μ M. Other useful BMP inhibitors include, but are not limited to, a 83-01; DMH-1; k02288; ML 347; SB 505124; and so on. SB-431542 is a TGF-beta inhibitor, and can be added at a concentration of at least about 0.1. mu.M, at least about 1. mu.M, at least about 5. mu.M, at least about 10. mu.M, at least about 50. mu.M, and at most about 100. mu.M, in an effective amount to inhibit TGF-beta signaling but not affect BMP signaling. Other useful TGF β inhibitors include, but are not limited to, LDN-193189 (journal of clinical research 2015, 125 (2): 796-; gallunertib (LY2157299) (cancer study 2014, 74 (21): 5963-77); LY2109761 (toxicology, 2014, 326C: 9-17); SB525334 (cell signaling, 2014, 26 (12): 3027-35); SD-208; EW-7197; kartogenin; DMH 1; LDN-212854; ML 347; LDN-193189HCl (Proc. Natl. Acad. Sci. USA, 2013, 110 (52): E5039-48); SB 505124; pirfenidone (histochemistry and cell biology, 2014, 10.1007/s 00418-014-1223-0); repsox; k02288; hesperetin; GW 788388; LY 364947; and so on. The medium containing the TGF inhibitor and BMP inhibitor may be changed daily.

An effective amount of a GSK-3 inhibitor may be included in the culture medium. For example, an effective dose of CHIR99021 may be added at a concentration of about 0.5 μ M to about 50 μ M, about 1 μ M to about 25 μ M, about 1 μ M to about 10 μ M, about 1 μ M to about 5 μ M, about 1 μ M to about 3 μ M, or the concentration of CHIR99021 may be about 1.5 μ M. Other useful GSK-3 inhibitors include, but are not limited to, CT98014, CT98023, CT99021, TWS119, SB-216763, SB-41528, AR-A014418, AZD-10806-BIO, Dibromanharelline, Hymenialdesine, indirubin, Meridianin, Alsterpaullone, Cazpaullone, Kenpaullone, and the like. The GSK-3 inhibitor may be added to the medium at the same time as the BMP inhibitor and TGF inhibitor, or may be added to the medium after about 1, 2, or 3 days of the addition of the BMP inhibitor and TGF inhibitor. For example, the medium may be supplemented with a GSK-3 inhibitor after 1 to 2 days (e.g., after 1 day) of incubation with the BMP inhibitor and TGF inhibitor.

The method may comprise culturing in the medium comprising the BMP inhibitor, TGF inhibitor, and GSK-3 inhibitor for at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, and up to about 10 days, up to about 9 days, up to about 8 days, up to about 7 days, up to about 6 days, up to about 5 days. For example, the neural induction step may comprise culturing for 1 to 2 days, e.g., 1 day, in a medium comprising a BMP inhibitor and a transforming growth factor beta (TGF β) inhibitor, supplementing the medium with a GSK-3 inhibitor, and culturing for 4 to 10 days, e.g., 7 days, in a medium comprising the BMP inhibitor, the TGF β inhibitor, and the GSK-3 inhibitor. The medium containing the TGF inhibitor, BMP inhibitor and GSK-3 inhibitor may be changed daily.

The BMP inhibitor concentration may be reduced during the culturing in the medium. For example, culturing in the medium comprising the BMP inhibitor, TGF inhibitor, and GSK-3 inhibitor can comprise (1) culturing for 2 to 5 days in a medium comprising the BMP inhibitor, TGF inhibitor, and GSK-3 inhibitor, wherein the TGF inhibitor is present at a concentration of about 5 μ Μ to about 20 μ Μ, about 5 μ Μ to about 15 μ Μ, about 8 μ Μ to about 12 μ Μ, or about 10 μ Μ; followed by (2) culturing for 2 to 5 days in a medium comprising the BMP inhibitor, a TGF inhibitor, and a GSK-3 inhibitor, wherein the TGF inhibitor is present at a concentration of about 1 μ M to about 5 μ M, about 2 μ M to about 4 μ M, or about 2.5 μ M.

The human midrib nucleus spheroids. After being placed in suspension culture for about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, the floating neural progenitor spheroids are transferred to neural medium to differentiate the neural progenitor cells. An exemplary neural medium is a medium comprising a neural basal medium, a vitamin a-free B-27 supplement, and a GlutaMAX supplement. Supplementing the neural medium with a GSK-3 inhibitor, a sonic hedgehog pathway agonist and FGF 4.

The GSK-3 inhibitor may be as described above. In certain embodiments, the neural medium is supplemented with, for example, a concentration of about 0.5 μ M to about 50 μ M, about 1 μ M to about 25 μ M, about 1 μ M to about 10 μ M, about 1 μ M to about 5 μ M, about 1 μ M to about 3 μ M, or CHIR99021 at a concentration of about 1.5 μ M.

Suitable sonic hedgehog pathway agonists include smooth agonists (SAG, CAS 364590-63-6) that modulate the coupling of Smo to its downstream effectors by interacting with the Smo heptahelical domain (K)_D59 nM). SAG may be provided in the neural medium at a concentration of about 10nM to about 1. mu.M, about 50nM to about 0.5. mu.M, about 75nM to about 0.25. mu.M, or at a concentration of about 100 nM.

In certain embodiments, the neural media is supplemented with FGF4, such as at a concentration of about 1ng/ml to about 100ng/ml, about 5ng/ml to about 50ng/ml, about 5ng/ml to about 15ng/ml, or about 10 ng/ml. The FGF4 can be added to the neural medium at the same time as the GSK-3 inhibitor and sonic hedgehog pathway agonist, or can be added to the neural medium after the BMP inhibitor and TGF β inhibitor are added for at least about 2 days, at least about 3 days, and up to about 10 days, up to about 7 days, up to about 6 days, or up to about 5 days. For example, the neural medium can be supplemented with FGF4 after 1 to 5 days (e.g., after 3 days) of culture in the neural medium containing the GSK-3 inhibitor and sonic hedgehog pathway agonist.

The step of differentiating the neural spheroid into an hnns may comprise culturing in the neural medium comprising the GSK-3 inhibitor and sonic hedgehog pathway agonist for at least about 2 days, at least about 3 days, and up to about 10 days, up to about 7 days, up to about 6 days, or up to about 5 days. For example, the step of differentiating the neural spheroid into an hnns may comprise culturing in a neural medium comprising the GSK-3 inhibitor and sonic hedgehog pathway agonist for 2 to 5 days (e.g., 3 days), supplementing the neural medium with FGF4, and culturing in a neural medium comprising the GSK-3 inhibitor, sonic hedgehog agonist, and FGF4 for at least 1 week, at least 2 weeks, at least 3 weeks, at most about 5 weeks, at most about 4 weeks, or 1 to 3 weeks. The medium containing the GSK-3 inhibitor, sonic hedgehog agonist and FGF4 can be changed daily.

As demonstrated by the examples, the use of a combination of a GSK-3 inhibitor, sonic hedgehog pathway agonist and FGF4 can form an hRNS with high levels of markers (indicative of the human nucleus raphe), e.g., at least 2 weeks after induction of suspension cultured hiPS cells to neural fates. For example, the hnns may have high levels of transcription factors that drive caudal midbrain/hindbrain development, such as NKX6-1, NKX2-2, OLIG2, GATA2, GATA3, LMX1B, FOXA2, EN1, but lower levels of forebrain markers, such as FOXG 1. Methods for determining the expression level of transcription factors include RT-qPCR as further described in the examples. In some embodiments, the methods disclosed herein further comprise determining whether the hRNS expresses a transcription factor that drives caudal mesencephalon/afterbrain development. An hRNS with high or low levels of transcription factor can have significantly higher or lower levels of gene expression compared to gene expression in a non-mesomeric nuclear spheroid, such as a cortical spheroid (hCS), when calculated using standard statistical tests.

To promote differentiation of neural progenitor cells to neurons, the neural medium is replaced to replace the GSK-3 inhibitor, sonic hedgehog pathway agonist with an effective dose of BDNF and NT3 about 1 week, about 2 weeks, about 3 weeks, about 4 weeks after the transfer of the neural spheroids to the neural medium. The growth factor may be provided at a respective concentration of at least about 0.5ng/ml, at least about 1ng/ml, at least about 5ng/ml, at least about 10ng/ml, at most about 500ng/ml, at most about 250ng/ml, at most about 100ng/ml, at most about 20ng/ml, or about 10 ng/ml.

The neural medium at this stage can typically be optionally supplemented with an effective dose of one or more of the following agents that promote neuronal activity: gamma secretase inhibitors, such as DAPT, at concentrations of about 1 to 25mM, about 2 to 10mM, and may be about 2.5 mM; l-ascorbic acid at a concentration of about 10 to 500nM, about 50 to 250nM, and may be about 200 nM; cAMP at a concentration of about 10 to 500nM, about 50 to 150nM, and may be about 100 nM; and docosahexaenoic acid (DHA) at a concentration of about 1 μ Μ to 100 μ Μ, about 5 μ Μ to about 50 μ Μ,5 μ Μ to 25 μ Μ, or may be about 10 μ Μ. In some embodiments, the neural medium comprises an effective dose of BDNF, NT3, a gamma secretase inhibitor, L-ascorbic acid, cAMP, and DHA.

To promote differentiation of neural progenitor cells into neurons, the neural spheroids can be cultured in neural medium comprising the factors listed above for at least about 1 week, at least about 2 weeks, at least about 3 weeks, up to about 6 weeks, up to about 5 weeks, up to about 4 weeks, about 1 to about 3 weeks, or about 2 weeks. The neural medium may further comprise FGF4 for at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at most about 10 days, at most about 8 days, at most about 6 days, about 2 to about 10 days, or about 5 days.

For example, the step of promoting differentiation of neural progenitor cells into neurons may comprise: (4) culturing the neural spheroids in suspension culture in a neural medium comprising FGF4 and at least one of the compounds selected from the group consisting of: brain Derived Neurotrophic Factor (BDNF), NT-3, L-ascorbic acid 2-trisodium phosphate (AA), N6, 2' -O-dibutyryladenosine 3',5' -cyclic monophosphate sodium salt (cAMP), cis-4, 7,10,13,16, 19-docosahexaenoic acid (DHA) and DAPT; and (5) culturing said neurospheres in suspension culture in a neural culture medium comprising said at least one compound in the absence of FGF4 for at least 1 week.

The spheroids may be maintained in neural culture for an extended period of time, e.g., 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months or longer, about 1 week, 2 weeks, 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks after the neural spheroids are transferred to the neural culture medium. In some embodiments, the spheroids are retained for 3 months or more. The spheroids may be maintained in neural medium in the absence of growth factors.

The hRNS comprises functional serotonergic neurons. As noted above, the majority of neurons originating from the human raphe nucleus are serotonergic neurons that project to multiple locations of the human central nervous system, including the human cortical regions. The presence of the serotonergic neurons can be detected by determining the expression of serotonin, an enzyme involved in the serotonin pathway (e.g., tryptophan 5-hydroxylase 2(TPH2)), and/or a marker of mature serotonergic neurons (e.g., vesicular monoamine transporter 2(VMAT2) and serotonin reuptake transporter (SERT)), such as using immunohistochemistry. The function of the neuron can be determined by monitoring neuron activity, e.g., on Ca²⁺And imaging the activity.

Human cortical spheroids. hCS can be produced by the methods described previously, for example as described in the following publications: pasca et al, (2015) nature: method, 12 (7): 671-678 entitled "functional cortical neurons and astrocytes from human pluripotent stem cells in 3D culture," the disclosure of which is incorporated herein by reference.

For example, as described above, hiPS cells in suspension culture are cultured to provide neural progenitor spheroids. After being placed in suspension culture for about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, the floating neural progenitor spheroids are transferred to neural medium to differentiate the neural progenitor cells. The medium is supplemented with an effective dose of FGF2 and EGF. The growth factor may be provided at a concentration of at least about 0.5ng/ml, at least about 1ng/ml, at least about 5ng/ml, at least about 10ng/ml, at least about 20ng/ml, at most about 500ng/ml, at most about 250ng/ml, at most about 100ng/ml, respectively.

To promote differentiation of neural progenitor cells to hCS comprising glutamatergic neurons, the neural medium was replaced to replace the FGF2 and EGF with effective doses of BDNF and NT3 about 1 week, about 2 weeks, about 3 weeks, about 4 weeks after FGF2/EGF exposure. The growth factor may be provided at a concentration of at least about 0.5ng/ml, at least about 1ng/ml, at least about 5ng/ml, at least about 10ng/ml, at least about 20ng/ml, at most about 500ng/ml, at most about 250ng/ml, at most about 100ng/ml, respectively. The cortical spheroids comprise functional glutamatergic neurons.

And (4) assembling the assembly. The hRNS can functionally integrate with human cortical spheroids (hCS) cultured alone to form cortical-mesomeric nuclear assemblies (hCS-hRNS) comprising glutamatergic and serotonergic neurons. The hCS-hRNS thus obtained contains the neural circuits located between the cortex and the raphe nucleus, and functionally integrates these circuits. For example, the functionally integrated cells interact in a physiologically relevant manner, e.g., forming synapses, transmitting signals, forming multicellular structures, etc.

Co-culturing the cortical spheroids with the human metakaryotic spheroids in neural medium under conditions that allow cell fusion. Conditions that allow cell fusion may include culturing the hnns and hCS in close proximity, e.g., in direct contact with each other.

After about 30 days, about 60 days, about 90 days of culture (applicable to hnns); after about 30 days, about 60 days, about 90 days of culture (for hCS), spheroids may be used for assembly. The hnns and hCS spheroids can be co-cultured for 2 days, 3 days, 5 days, 8 days, 10 days, 14 days, 18 days, 21 days, or longer. Assembly can be performed in neural media. The resulting cortical-raphocene spheroid assembly is shown to contain a functional neural circuit, wherein the spheroid assembly comprises a bi-directional projection between the cortex and the raphocene spheroid, and the serotonergic neurons of the raphocene spheroid are capable of modulating the activity of the cortical neural circuit. Methods for confirming the function of the neuron are known in the art and include optogenetic methods and imaging of calcium activity in neurons, such as the methods described in the examples. In some embodiments, the method may comprise confirming the function of a neuron in the cortical-raphocene class assembly.

Screening assays

Also disclosed herein are screening assays that involve determining the effect of a candidate agent on the spheroid (e.g., hnns), assembloid (e.g., hCS-hnns), or cell derived therefrom. The candidate agent may be a small molecule or a genetic element. The screening assay may involve contacting the candidate agent with the spheroid, class assembly, or a cell derived therefrom, and determining the effect of the candidate agent on a parameter of the spheroid, class assembly, or cell, wherein such parameter includes a change in morphology, gene, or function.

For example, a screening assay may involve determining the effect of a candidate agent (e.g., an SSRI inhibitor) on the function of a neural circuit in a spheroid or class assembly. As described herein, it was demonstrated that the hCS-hRNS-like assembly comprises neurons projecting bi-directionally between hCS-hRNS, and that the serotonergic neurons of hRNS are capable of modulating the function of cortical neural circuits as revealed by the combination of viral markers and calcium imaging with light stimulation. Thus, the screening assay may involve determining whether a candidate agent is capable of altering the ability of serotonergic neurons to modulate the function of the cortical neural circuit in the hCS.

In addition, as described herein, various diseases and disorders are associated with serotonergic dysfunction. Thus, the assays described herein may be particularly useful where the spheroid or spheroid-like assembly comprises at least one allele associated with a neurological or psychiatric disorder, schizophrenia, affective disorder (e.g., MDD, bipolar disorder, or anxiety disorder), and Autism Spectrum Disorder (ASD). Candidate agents that are capable of restoring function, such as to neural circuits (e.g., cortical neural circuits) in spheroids or similar assemblies comprising alleles associated with these disorders, may have therapeutic utility in the treatment of the disorder.

Furthermore, the class of assemblies described herein can be used to dissect cell autonomous contributions in these disorders. For example, an assembly-like body can be generated in which one spheroid (e.g., hnns or hCS) is derived from a patient having a disorder described herein, and the other spheroid is derived from an unaffected individual, i.e., a subject not having the same disorder. For example, in a method of generating an assembly-like body from a first and a second human pluripotent stem cell, the first or second human pluripotent stem cell may comprise at least one allele associated with a neurological or psychiatric disorder.

Neural activity causes rapid changes in intracellular free calcium. Therefore, calcium imaging assays that utilize this can be used to determine the function of neuronal circuits. This may involve modifying neurons to contain the gene-encoded calcium indicator protein so that the protein includes the fluorophore sensor GCaMP and imaging these cells. GCaMP comprises a circulating array of green fluorescent protein, calcium binding protein calmodulin (CaM), and M13 peptide that interacts with CaM, wherein the brightness of GFP increases upon calcium binding. For further details on calcium imaging assays see the following publications: chen et al, (2013) nature, 499 (7458): 295-300. Other calcium imaging assays include Fura-2 calcium imaging, Fluo-4 calcium imaging, and Cal-590 calcium imaging.

For example, the neuron may be modified to express GCamP6 f. This may be combined with methods that activate certain neurons in response to external stimuli, such as optogenetic methods that activate neurons in response to light. For example, to test function between two neurons involved in a neural circuit, a "first" neuron may be modified to express a optogenetic actuator (e.g., chrimson r), and a "second" neuron may be modified to express a calcium indicator (e.g., GCamP6f) and used for imaging to monitor calcium release. If the first neuron is functionally connected (synaptically connected) to the second neuron, then optogenetic activation of the first neuron will affect intracellular calcium levels as well as the visible readout of the second neuron.

As mentioned above, serotonin can elicit a variety of different responses, which may depend on the receptor on which serotonin acts. Thus, a method of determining the ability of serotonergic neurons to modulate cortical neural circuits may comprise labeling cells of the hRNS with a optogenetic actuator (e.g., chrimson r), labeling cells of the hCS with a calcium indicator, stimulating cells of the hRNS in the hRNS-hCS-like assemblies, and determining whether calcium activity is increased or decreased in the cells of the hCS in the like assemblies. Such increase or decrease may be transient or may occur throughout the post-stimulation period. As exemplified herein, such methods are used to confirm serotonergic modulation of cortical neural circuits in the hRNS-hCS-like assemblies. To determine the effect of a candidate agent on such serotonergic modulation, the optogenetic method can be performed in the presence and absence of the candidate agent and comparing the results in both conditions.

Also of interest are analytical methods at the single cell level, for example as described above: real-time imaging (including confocal or light-sheet microscopy), single cell gene expression or single cell RNA sequencing, calcium imaging, immunocytochemistry, patch-clamping, flow cytometry, and the like. Various parameters can be measured to determine the effect of a drug or treatment on the spheroid, class assembly or cells derived therefrom. For example, single cell RNA sequencing of the cells that make up the spheroid or class of assemblies can be used to characterize the characteristics of these cells, and in assays aimed at determining whether a candidate agent affects cell fate.

The parameter is a quantifiable component of the cell, particularly one that can be accurately measured in a high-throughput system. The parameter may also be any cellular component or cellular product, including a cell surface determinant, receptor, protein or conformational or post-translational modification thereof, lipid, carbohydrate, organic or inorganic molecule, nucleic acid (e.g., mRNA, DNA, etc.), or a portion derived from such a cellular component, or a combination thereof. While most parameters will provide quantitative readings, in some cases semi-quantitative or qualitative results are acceptable. The reading may comprise a single determined value, and may also comprise a mean, median or variance, etc. The respective values are expected to vary and the range of values for each parameter in the set of test parameters is obtained using standard statistical methods and conventional statistical methods for providing individual values.

Parameters of interest include detection of cytoplasmic, cell surface or secreted biomolecules, biopolymers, such as polypeptides, polysaccharides, polynucleotides, lipids, and the like. Cell surface and secreted molecules are preferred types of parameters because they mediate cellular communication and cellular effector responses and can be more easily measured. In one embodiment, the parameter comprises a specific epitope. Epitopes are often identified using specific monoclonal antibodies or receptor probes. In some cases, the molecular entity comprising the epitope is from two or more species and comprises a defined structure; examples include epitopes associated with heterodimeric integrins determined by combinatorial methods. The parameter may be the detection of a specifically modified protein or oligosaccharide. The parameters may be defined by specific monoclonal antibodies or ligands or receptor binding determinants.

Candidate agents of interest are bioactive agents that encompass many chemical classes, primarily organic molecules, which may include organometallic molecules, inorganic molecules, gene sequences, and the like. Important aspects of the invention are the evaluation of drug candidates, the selection of therapeutic antibodies with preferred biological response functions and protein-based therapeutics. Candidate agents contain functional groups necessary for structural interaction (especially hydrogen bonding) with proteins and typically include at least one amino, carbonyl, hydroxyl, or carboxyl group, often at least two functional chemical groups. The candidate agents typically comprise cyclic carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups. Candidate agents may also be derived from biomolecules including peptides, polynucleotides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs, or combinations thereof.

It also includes pharmacologically active drugs, genetically active molecules, and the like. Compounds of interest include chemotherapeutic agents, anti-inflammatory agents, hormones or hormone antagonists, ion channel modifiers, and neuroactive agents. Examples of pharmaceutical formulations suitable for use in the present invention are those described in the following publications: "pharmacological basis for therapeutics", Goodman and Gilman, McGraw-Hill, new york city, new york, (1996), ninth edition, the following sections: drugs acting on synapses and at the junction of neuroeffectors; a cardiovascular agent; vitamins, dermatology; and toxicology, all of which are incorporated herein by reference.

An important class of candidate agents for use in conjunction with the compositions and methods described herein are Selective Serotonin Reuptake Inhibitors (SSRIs). SSRIs are a class of drugs commonly used as antidepressants for the treatment of Major Depressive Disorder (MDD) and anxiety disorders. SSRIs typically act by increasing extracellular serotonin levels by limiting serotonin reuptake. Examples of known SSRIs include citalopram, escitalopram, fluoxetine, fluvoxamine, paroxetine, sertraline, dapoxetine. In addition to investigating the role of these known SSRIs, the system described herein can also be used as part of a screening assay to discover new SSRIs.

The test compound includes all types of molecules described above, and may further comprise an unknown amount of the sample. Of interest are complex mixtures of naturally occurring compounds derived from natural sources (e.g., plants). While many samples contain solutions of compounds, solid samples that are soluble in a suitable solvent can also be determined. Samples of interest include environmental samples such as groundwater, seawater, mining waste, and the like; biological samples, such as lysates prepared from crops, tissue samples, and the like; manufacturing samples, such as time courses in drug preparation processes; and a library of compounds prepared for analysis; and so on. Samples of interest include compounds that are being evaluated for potential therapeutic value, i.e., drug candidates.

The term sample also includes the above-mentioned fluids to which additional components have been added, such as components that affect ionic strength, pH, total protein concentration, and the like. In addition, the sample may be processed to effect at least partial fractionation or concentration. If care is taken to reduce degradation of the compound, the biological sample may be stored under nitrogen, stored frozen, a combination thereof, or the like. The volume of sample used is sufficient for measurable detection, usually about 0.1 to 1ml of biological sample is sufficient.

Compounds, including candidate agents, can be obtained from a variety of sources, including libraries of synthetic or natural compounds. For example, a number of approaches are available for the random and directed synthesis of various organic compounds (including biomolecules), including the expression of random oligonucleotides and oligopeptides. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts can be obtained or readily produced. In addition, natural or synthetically produced libraries and compounds can be readily modified by conventional chemical, physical and biochemical means and can be used to generate combinatorial libraries. Known agents may be chemically modified (e.g., acylated, alkylated, esterified, amidated, etc.) directly or randomly to produce structural analogs.

The term "genetic element" as used herein refers to polynucleotides and analogs thereof, wherein the genetic element is tested by adding the genetic element to a cell in the screening assays of the invention. The introduction of the genetic factor results in an alteration of the overall genetic composition of the cell. Genetic elements (e.g., DNA) can cause experimentally-introduced changes to the cellular genome, typically by integrating the sequences into the chromosome, e.g., using CRISPR-mediated genome engineering (see, e.g., Shmakov et al, (2017) natural review: microbiology, 15: 169). The genetic change may also be a transient change in which the exogenous sequence is not integrated, but rather remains as an episome. Genetic factors (e.g., antisense oligonucleotides) can also affect the expression of proteins without changing the genotype of the cell by interfering with the transcription or translation of mRNA. The effect of the genetic element is to increase or decrease the expression of one or more gene products in the cell.

The introduction of an expression vector encoding a polypeptide can be used to express the encoded product in a cell lacking the sequence, or to overexpress the product. Various constitutive or externally regulated promoters can be used, wherein in the latter case gene transcription can be switched on or off. These coding sequences may include full length cDNA or genomic clones, fragments derived therefrom, or chimeras combining naturally occurring sequences with other coding sequence functions or domains. Alternatively, the introduced sequence may encode an antisense sequence; is an antisense oligonucleotide; RNAi, encoding a dominant negative mutation, or a dominant or constitutively active mutation of the native sequence; altered regulatory sequences, and the like. The expression vector may be a viral vector, such as an adeno-associated virus, adenovirus, herpes simplex virus, retrovirus, lentivirus, alphavirus, flavivirus, rhabdovirus, measles virus, newcastle disease virus, poxvirus, and picornavirus vector.

Antisense and RNAi oligonucleotides can be chemically synthesized by methods known in the art. Preferred oligonucleotides are chemically modified with their own phosphodiester structure to increase their intracellular stability and binding affinity. Many such modifications have been described in the literature which alter the chemical nature of the backbone, sugar or heterocyclic base. Useful variations in backbone chemistry are phosphorothioates, phosphorodithioates in which both non-bridging oxygens are substituted with sulfur, phosphoramidites, alkylphosphotriesters, and boronic acid phosphates. The achiral phosphate derivatives include 3' -O ' -5' -S-thiophosphate, 3' -S-5' -O-thiophosphate, 3' -CH2-5' -O-phosphonate and 3' -NH-5' -O-phosphoramide. Peptide nucleic acids replace the entire ribose-phosphodiester backbone with peptide bonds. Sugar modifications are also used to enhance stability and affinity, for example morpholino oligonucleotide analogs.

Multiple assays are performed in parallel with different concentrations of the agent to obtain different responses to various concentrations. As is well known in the art, determining an effective concentration of an agent typically uses a concentration range derived from a 1:10 or other logarithmic dilution. If necessary, a second series of dilutions can be used to further refine the concentration. Typically, one of these concentrations is used as a negative control, i.e., zero concentration or below the detection level of the agent, or at or below the concentration of the agent at which no detectable phenotypic change occurs.

In addition to the functional parameters described above, various methods may be utilized to quantify the presence of selected parameters. For measuring the amount of molecule present, a suitable method is to label the molecule with a detectable moiety, which may be fluorescent, luminescent, radioactive, enzymatically active, etc., especially a molecule that specifically binds to the parameter with high affinity, the fluorescent moiety being readily available for labeling virtually any biomolecule, structure or cell type. The immunofluorescence part can be combined with specific protein, and can also be combined with site modification such as specific conformation, cleavage products or phosphorylation. Individual peptides and proteins can be engineered to fluoresce, for example, by expressing them as intracellular green fluorescent protein chimeras (for review, see Jones et al, (1999) Biotechnology trends, 17 (12): 477-81). Thus, antibodies can be genetically modified to provide fluorescent dyes as part of their structure

Depending on the label chosen, parameters may be measured using methods other than fluorescent labeling, immunoassay techniques (e.g., Radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA), homogeneous enzyme immunoassay), and related non-enzymatic techniques. These techniques utilize specific antibodies as reporter molecules, which are particularly useful due to their high specificity for attachment to a single molecular target. Us patent No. 4,568,649 describes a ligand detection system that employs scintillation counting. These techniques are particularly useful for proteins or modified protein parameters or epitopes or carbohydrate determinants. Cellular readings for proteins and other cellular determinants can be obtained using fluorescent or other labeled reporter molecules. Cell-based ELISA or related non-enzymatic or fluorescence-based methods are capable of measuring cell surface parameters and secretion parameters. The Capture ELISA and related non-enzymatic methods typically employ two specific antibodies or reporter molecules that can be used to measure parameters in solution. Flow cytometry methods can be used to measure cell surface and intracellular parameters, as well as shape change and particle size, and can be used to analyze magnetic beads for use as antibodies or probe-linking reagents. The readout for such an assay may be the average of the fluorescence associated with the detection of a cell surface molecule or cytokine by fluorescent antibody alone, or the mean fluorescence intensity, the median fluorescence intensity, the change in fluorescence intensity, or some relationship between these values.

The art uses single cell multiparameter and multicellular multiparameter multiplex assays where input cell types are identified and the parameters are read by quantitative imaging and fluorescence and confocal microscopy, see confocal microscopy and protocols for details (molecular biology methods, volume 122). Paddock, eds., Humana Press, 1998. See U.S. patent No. 5,989,833 issued on 23/11 1999.

The assay results may be input to a data processor to provide a data set. Algorithms are used to compare and analyze data obtained under different conditions. Factors and the effect of the medicament are read by determining the variation of a plurality of parameters. The data will include results from combinations of assays performed with the agents, and may also include one or more of control status, simulated status, and results from other combinations of assays performed with other agents or under other conditions. The results may be presented visually in a chart for quick and easy comparison, and may include numbers, charts, color representations, and the like.

The data set is compiled from values obtained by: the parameter is measured in the presence and absence of different cells (e.g., genetically modified cells, cells cultured in the presence of specific factors or agents that affect neuronal function), and the presence of the agent of interest is compared to at least one other state, typically a control state, which may include a state without the agent or with a different agent. The parameters include functional states such as synapse formation and calcium ions in response to a stimulus, the levels of which may vary in the presence of the factor. Ideally, the results are normalized with respect to a standard (typically a "control value or state") to provide a normalized data set. The values obtained from the test conditions can be normalized by: the unstimulated control value is subtracted from the measured value and the corrected measured value is divided by the corrected stimulated control value. Other normalization methods may also be used; and a log or other derivative of the measured value or a ratio of the measured value to the stimulus value or other control value may be used. Under control conditions, the data is normalized to control data for the same cell type, but the data set may comprise normalized data from one, two or more cell types and assay conditions.

The data set may include level values for sets of parameters obtained under different assay combinations. It is programmed to provide values for a sufficient number of alternative assay combinations to allow compilation of comparison values.

The database may be compiled based on the experimental set, for example, the database may contain data obtained from a set of assay combinations having a plurality of different environmental changes, where each change may be a series of related compounds or may be a compound representing a different class of molecules.

A mathematical system can be used to compare data sets and quantitatively measure similarities and differences between them. For example, the data set may be analyzed by pattern recognition algorithms or clustering methods (e.g., hierarchical or k-means clustering, etc.) that use statistical analysis (correlation coefficients, etc.) to quantify the correlation. These methods may be modified (by weighting, employing classification strategies, etc.) to optimize the ability of the data set to distinguish between different functional effects. For example, as the data set is analyzed, more or less weight may be given to various parameters to enhance the discriminative power of the analysis. The effect of varying the weight assigned to each parameter is evaluated and an iterative process is used to optimize the pathway or cell function differentiation.

Comparison of the data set obtained from the test compound with the reference data set is accomplished using a suitable derivation scheme, AI system, statistical comparison, and the like. Preferably, the data set is compared to a database of reference data. Similarity to reference data involving known pathway stimulators or inhibitors may provide an initial indication of cellular pathways targeted or altered by the subject stimuli or agents.

A reference database may be compiled. These databases may include reference data from a kit comprising known agents or combinations of agents targeting a particular pathway, as well as reference data from analysis of cells processed under environmental conditions, where single or multiple environmental conditions or parameters are deleted or specifically altered. Reference data can also be generated based on a kit containing cells with genetic constructs that selectively target or modulate a particular cellular pathway. In this way, a database is developed that can reveal the contribution of individual pathways to complex reactions.

The effectiveness of the pattern search algorithm in classification may involve optimization of the number of parameters and assay combinations. The disclosed techniques for selecting parameters provide the computational requirements to derive physiologically relevant outputs. In addition, these techniques of pre-filtering the data set (or potential data set) using cellular activity and disease-related biological information increase the likelihood that the output retrieved back from the database is relevant to predicting the mechanism of the agent and the effect of the agent in vivo.

To develop an expert system for the selection and classification of biologically active pharmaceutical compounds or other interventions, the following procedure was adopted. For each reference and test pattern, a data matrix is typically generated, where each point in the data matrix corresponds to a reading of a parameter, where the data for each parameter may be from repeated assays, e.g., multiple individual cells of the same type. As previously mentioned, data points may be quantitative, semi-quantitative, or qualitative, depending on the nature of the parameter.

The reading may be a mean, average, median, variance, or other statistically or mathematically derived value associated with the measurement. The parameter reading information can be further refined by directly comparing the corresponding reference readings. The absolute values obtained for each parameter under the same conditions will show the inherent variability of the living organism and may also reflect the individual cell variability as well as the inherent variability between individuals.

The classification rules are constructed based on a training data set (i.e., a data matrix) obtained from a number of repeated experiments. The classification rules are chosen so as to correctly identify the repetitive reference patterns and successfully distinguish between the different reference patterns. The classification rule learning algorithm may include a decision tree method, a statistical method, a naive bayes algorithm, and the like.

The knowledge database will be of sufficient complexity to efficiently identify and classify new data under test. Several methods for generating a set of well-covered classification patterns and sufficiently powerful mathematical/statistical methods for distinguishing them can achieve this.

Data from cells treated with specific drugs known to interact with a particular target or pathway provides a more detailed set of sorting readings. Data generated based on cells genetically modified using overexpression and antisense technologies allows testing of the impact of individual genes on phenotype.

The preferred knowledge database contains reference data from the optimized cells, environment and parameter set. For complex environments, the knowledge database may also include data reflecting minor changes in the environment, such as environments in which one or more factors or cell types of interest are excluded or included or quantitatively changed, such as concentrations or exposure times, and the like

For further elucidation of the general techniques useful in practicing the present invention, practitioners may refer to standard textbooks and reviews of cell biology, tissue culture, embryology, and neurobiology. For tissue culture and embryonic stem cells, the reader is not referred to teratocarcinoma and embryonic stem cells: one practical approach (e.j.robertson, editors, IRL Press ltd., 1987); technical guidelines for mouse development (p.m. wasserman et al, editors, Academic Press, 1993); embryonic stem cell differentiation in vitro (m.v. wiles, methods in enzymology, 225: 900, 1993); characteristics and uses of embryonic stem cells: application to human biology and gene therapy (p.d. rathjen et al, reproduction, fertility and development, 10: 31, 1998).

General methods of molecular and cellular biochemistry are described in the following standard texts: molecular cloning: a Laboratory Manual, 3 rd edition (Sambrook et al, Harbor Laboratory Press 2001); a molecular biology laboratory Manual, 4 th edition (eds: Ausubel et al, John Wiley & Sons 1999); protein method (Bollag et al, John Wiley & Sons 1996); non-viral vectors for gene therapy (eds.: Wagner et al, Academic Press 1999); viral vectors (edit: Kaplift and Loewy, Academic Press 1995); a manual for immunological methods (eds.: I.Lefkovits, Academic Press 1997); cell and tissue culture: biotech laboratory procedures (Doyle and Griffiths, John Wiley & Sons 1998). The reagents, cloning vectors and kits for gene manipulation referred to in the present invention are available from commercial suppliers, e.g., BioRad, Stratagene, Invitrogen, Sigma-Aldrich and Clontech.

Each publication cited in this specification is incorporated herein by reference in its entirety for all purposes.

The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of methods of making and using the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.) but some experimental error and deviation should be accounted for. Unless otherwise indicated, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees celsius, and pressure is at or near atmospheric.

Experiment of the invention

The present invention describes a novel method of using human pluripotent stem cells (hpscs) to study a functional neuromodulation system to generate three-dimensional (3D) assemblies containing forebrain organoids coupled to organoids mimicking the midriff and capable of transmitting serotonergic projections. We first generated hPSC-derived mid-gap nuclear spheroids (hRNS), which include functional serotonergic neurons as well as other resident cell types of the mid-gap nucleus. Human cerebral cortical spheroids (hCS), including pyramidal glutamatergic neurons of various cortical layers, were assembled with hnns to give hnns-hCS class assemblies in which there was a bidirectional projection between the cortex and the raphe nucleus. We combined single cell RNA sequencing, viral labeling and calcium imaging with light stimulation, demonstrated an in vitro human cell model of cortical circuit serotonergic regulation that can be used as a platform for understanding the assembly of these circuits, modeling MDD, autism spectrum disorders, and schizophrenia, and drug screening to identify better drugs targeting this neuromodulation pathway.

Example 1

Method

Production of human centronuclear spheroids (hRNS). Human pluripotent stem cells (hpscs) were plated on six-well plates with recombinant human vitronectin (VTN-N, Life Technologies, a14700) in Essential 8 medium (Life Technologies, a1517001) supplemented with penicillin and streptomycin (1:100, Gibco, 15140122). To generate hRNS, the hipSC was incubated with Accutase (Innovate Cell Technologies, AT-104) AT 37 ℃ for 7 minutes and dissociated into single cells. Approximately 300 million single cells were added to each AggreWell 800(STEMCELL Technologies, 34815) well in Essential 8 medium supplemented with ROCK inhibitor Y-27632 (10. mu.M, Selleckchem, S1049), centrifuged at 100g for 3 minutes to capture the cells in the microwells, and incubated at 37 ℃ and 5% CO 2. After 24 hours, we collected spheroids from each microwell by stably moving the medium up and down the wells (with the cut ends of the P1000 tips) and transferring it to an ultra-low adhesion plastic petri dish (Corning, 3262) supplemented with Essential 6 medium (Life Technologies, a1516401) in doxorphine (2.5 μ M, Sigma-Aldrich, P5499) and SB-431542(10 μ M, Tocris, 1614). From day 2 to day 8, the medium was changed daily and supplemented with doxorphine, SB-431542 (day 2 to day 5: 10. mu.M, day 5 to day 8: 2.5. mu.M) and the GSK-3 inhibitor CHIR99021 (1.5. mu.M).

On day 6, the neurospheres were transferred to neural media containing Neurobasal a (Life Technologies, 10888), vitamin a-free B-27 supplement (Life Technologies, 12587), GlutaMax (1:100, Life Technologies, 35050), and penicillin and streptomycin. From day 5 to day 15, the nerve medium was changed and supplemented with CHIR99021 (1.5 μ M) and the smooth agonist SAG (100nM) daily. From day 8 to day 20, the neural medium was also supplemented with fibroblast growth factor 4(FGF4, 10 ng/mL).

To promote differentiation, the neural medium was changed every other day from day 16 to day 30 and supplemented with BDNF (10ng/mL), NT-3(10ng/mL), IGF-1(10ng/mL), cAMP (100nM), L-ascorbic acid (200. mu.M) and docosahexaenoic acid (DHA, 10. mu.M). A schematic diagram illustrating different formulations is shown in fig. 1A. To characterize cellular diversity in hnns, we performed single cell transcriptomic manipulations of dissociated hnns at day 42 according to the supplier's recommendations (10x genomics, 120262). To quantify serotonin release in hRNS, 2-3 whole hRNS/time point/hiPS cell line were flash frozen at different time points and processed accordingly for High Performance Liquid Chromatography (HPLC). For the serotonin uncaging experiment, NPEC was used to lock serotonin (Tocris, 3991) to a final concentration of 50 μ M. The FRAP module of Leica SP8 confocal microscope was used to achieve glutamate uncalcining using UV light (405 nm).

Production of cortical-synuclein assemblies (hCS-hRNS). (FIG. 1A) to generate the cortical-mesocortical-mesonuclear assemblies (hCS-hRNS), hCS and hRNS were generated separately and then assembled by placing them next to each other in 1.5ml microcentrifuge tubes in an incubator for 3 days. The neural medium used for assembly contained neurobasal-A, vitamin A-free B-27 supplement, GlutaMax (1:100), penicillin and streptomycin (1: 100). On day 2, the medium was carefully changed. On day 3, the assembly-like bodies were placed in 24-well ultra low adhesion plates in the above described neural medium using a dissected P1000 pipette tip. Thereafter, the medium was changed every 3 to 4 days. hCS was produced using methods 13,14 as described previously. Assembly was performed between day 45 and day 60. For some experiments, hCS or hRNS were virus-tagged with AAV-DJ1-hSyn1 YFP seven to ten days prior to assembly. For the optogenetic photostimulation experiments, AAV1-hSyn1: Chrimson R-tdTomato virus was used to label hRNS and was assembled with the aforementioned hCS expressing EF1a-GCaMP 6.

Example 2

Generation of functional hRNS

To elaborate the midriff-like spheroids (organoids), hpscs accumulated in microwells were first patterned by dual SMAD inhibition of neuroectoderm, and then exposed to CHIR, SHH agonist SAG, and FGF4 (fig. 1B). Analysis of gene and protein expression by RT-qPCR and immunocytochemistry at day 15 post-patterning showed up-regulation of transcription factors driving caudal midbrain/hindbrain development (NKX6-1, NKX2-2, OLIG2, GATA2, GATA3, LMX1B, FOXA2, EN 1; fig. 1C, D) and down-regulation of forebrain marker FOXG1 (fig. 1C).

Day 52 immunocytochemistry revealed the presence of serotonergic neurons characterized by 5-hydroxytryptamine (serotonin, 5-HT) and tryptophan 5-hydroxylase 2(TPH2), one of the major enzymes in the serotonin synthesis pathway. The core molecular phenotype of mature serotonergic neurons includes vesicular monoamine transporter 2(VMAT2), which packages 5-HT into synaptic vesicles and the serotonin reuptake transporter SERT, and recovers extracellular 5-HT 15. Immunocytochemistry at day 52 revealed cells positive for both SERT and VMAT2 in hRNS (fig. 1E). Next, we used HPLC to measure 5-HT release in hRNS. In three different time points and cell lines, we consistently measured 5-HT in hRNS, ranging from 20-175ng/mL/mg protein, while for hCS we did not detect 5-HT at any time point, indicating the specificity of hRNS patterning for the sewing nucleus (FIG. 1F). Next, the gene expression profiles of eleven 5HT metabotropic receptors (HTR) in hCS were studied. We observed that a combination of excitatory Go/Gs/G11 couplings (pink, HTR2a, HTR6, HTR2c) and inhibitory Gi/Go couplings (green, HTR1b, 1d) was expressed in hCS at day 100 (fig. 1G).

To explore the diversity of cell types in hRNS, we performed single-cell transcriptomics manipulations of hRNS from day 79 to day 82. Unsupervised clustering of hnns cells revealed massive expression of postencephalic lineage neurons of typical markers among 5HT lineage markers ("5 HT neurons") as well as other neuronal subtypes ("gabanergic neurons", "glutamatergic neurons") (fig. 2A-B). A more careful examination of the 5-HT clustering revealed tail and rostral subgroups (FIG. 2C)

Example 3

Assembly of hCS-hRNS

To mimic the development and function of the cortico-internuclear loop, hRNS was virally labeled with AAV-DJ1-hSYN1 from day 45 to day 60 with mCherry and assembled with hCS 7-8 days later to give hRNS-hCS class assemblies. Real-time imaging of intact hRNS-hCS 16 days post-assembly (days post-fusion; daf) demonstrated hRNS-derived mCherry projected extensively into hCS⁺Cells (Picture)3A) In that respect Immunocytochemistry also demonstrated TPH2 projection into hCS⁺Cells (fig. 3B). To assess the directionality of the projections in hRNS-hCS, we virus-tagged hRNS and hCS with AAV-DJ1-hSYN1:: eYFP and AAV-DJ1-hSYN1:: mCherry, respectively, and then assembled them. Real-time imaging of the complete hrNS-hCS 50 days after assembly revealed bi-directional projections between the hrNS and hCS. Forebrain-projecting serotonergic cells in the nucleus raphanus exhibit different axonal morphologies with large oval varicose veins along the fine axons [4]. eYFP derived along hRNS in hCS⁺Similar structures were observed for the projected axons, whereas similar structures were not observed in the axons projected along the hCS-derived mCherry + (figure 3C).

Example 4

Functional detection of neuromodulation connectivity in hCS-hRNS

To label 5HT lineage neurons in hRNS for functional studies, we used viral reporter genes that drive emGFP expression under the FEV mini-promoter Ple67(AAV-Ple67:: emGFP). We characterized their specificity using immunostaining of dissociated hRNS cells infected with emGFP, Ple67:, with 5-HT and TPH2 (FIG. 4A); this experiment shows 80-90% emGFP⁺Is 5HT⁺Or TPH2⁺Indicating high specificity. Next, we used the iCRE-dependent version of the reporter (AAV-DJ-Ple67iCRE) and co-infected hRNS with AAV-EF1 α -DIO-eYFP to induce recombination while driving eYFP expression in cells of the 5-HT lineage. Then, we assembled the co-infected hRNS with hCS infected with AAV-hSYN1:: mCherry. The resulting hCS-hRNS showed extensive eYFP of 5-HT lineage cells from hRNS to hCS⁺Projected (fig. 4B). To functionally probe for serotonergic import of hCS in hCS-hRNS, we used the same iCRE-dependent Ple67 reporter gene in hRNS (AAV-Ple67iCRE and AAV-EF1 α -DIO-ChRmine-K_v2.1) expressing ChRmine-KV2.1, which is a somatically targeted red-shifted opsin, and assembling it with hCS labeled with a gene encoding a calcium indicator (AAV-hSYN1-GCamP7 s). Photostimulation of 5-HT lineage cells by high frequency photostimulation at 625nm wavelength reliably elicited hCS neuronal responses as shown by stimulus-locked calcium responses(FIG. 4C).

Citations

Neuroregulation of Nadim, F. and Bucher, D. neurons and synapses. New neurobiology, 0, 48-56 (2014), Bucher, d, and Marder, e, snapshot: and (4) nerve regulation. Role of cells 155, 482-482.e1(2013). Vitalis, t. and Parnavelas, j.g. serotonin in early cortical development. And (5) developing neuroscience. 25, 245-256 (2003) Hornung, j. -p. human nuclear and serotonergic systems. Journal of chemical neuroanatomy 26, 331-343 (2003), Jacobs, b.l. and Azmitia, e.c. the structure and function of the brain serotonin system. Physiological review. 72, 165-229 (1992). Hodge, R.D. et al. Conserved cell types with different characteristics between human and mouse cortex. bioRxiv 384826(2018) doi 10.1101/384826

E. And the like. The neurochemical differentiation of monoaminergic neurons of the human medullary spinal cord in the early stages of pregnancy. Brain study during development. 75,1-12(1993). Takahash et al. The distribution of serotonin-containing cell bodies in the brainstem of a human fetus was determined by immunohistochemistry using an antiserum against serotonin. Brain development 8, 355-365 (1986) Adell, A.review the role of suture and serotonin in neuropsychiatric disease. Journal of common physiology 145, 257-259 (2015), Sodhi, M.S.K. and Sanders-Bush, E.serotonin and brain development International neurobiology overview 59, 111-174 (Academic Press, 2004). Bonnin, a. et al. Nature 472, 347 one 350(2011), whittaker-Azmitia, p.m. serotonin and brain development: in human developmental diseases. Notification of brain research 56, 479-.

Functional cortical neurons and astrocytes from human pluripotent stem cells in a.m. et al.3D culture. Naturally: method 12, 671-678 (2015) Birey, F, et al. Nature 545, 54-59 (2017). okatty, b.w., Commons, k.g., and Dymecki, s.m. embrace the diversity of the 5-HT neuronal system. For a natural review: neuroscience.1 (2019). doi:10.1038/s41583-019-0151-3.Celada, p., Puig, m.v. and Artigas, f. cortical neurons and serotonergic regulation of the network. And (7) integrating recognition and treatment of neuroscience frontier.7, (2013) Frank, C. Release of 5-HT from nucleus accumbens by canadian family doctor 54, 988-. Nature 560,589(2018) Doan, r.n. et al. Naturally: genetics.1 (2019). doi:10.1038/s 41588-019-0433-8.

Claims (48)

1. A method for producing human metakaryotic spheroids or organoids (hRNS) in vitro, the method comprising:

(a) inducing human pluripotent stem cells in 3D suspension culture to a neural fate to generate neurospheres;

(b) differentiating said neural spheroids into hRNS; and

(c) maintaining the hRNS in neural culture medium such that the hRNS comprises serotonergic neurons.

2. The method of claim 1, wherein the human pluripotent stem cell is an induced human pluripotent stem cell.

3. The method of claim 1, wherein inducing the human pluripotent stem cells into the neural fate in suspension culture in step (a) comprises culturing in a culture medium comprising a Bone Morphogenetic Protein (BMP) inhibitor and a transforming growth factor beta (TGF β) inhibitor, and wherein the method comprises supplementing the culture medium with a GSK-3 inhibitor.

4. The method of claim 3, wherein the BMP inhibitor is doxorphine and the TGF β inhibitor is SB-431542.

5. The method of claim 3 or claim 4, wherein the GSK-3 inhibitor is CHIR 99021.

6. The method of claim 3, wherein the medium comprising the BMP inhibitor and TGF β inhibitor is cultured for 2 to 10 days.

7. The method of claim 6, wherein the medium is supplemented with a GSK-3 inhibitor after 1 to 2 days, the medium comprising the BMP inhibitor, TGF inhibitor, and GSK-3 inhibitor, optionally wherein the medium comprising the BMP inhibitor, TGF inhibitor, and GSK-3 inhibitor is cultured for 4 to 8 days.

8. The method of claim 7, wherein culturing in the medium comprising the BMP inhibitor, TGF inhibitor, and GSK-3 inhibitor comprises:

(1) culturing for 2 to 5 days in a medium comprising the BMP inhibitor, a TGF inhibitor, and a GSK-3 inhibitor, wherein the TGF inhibitor is present at a concentration of 5 μ M to 20 μ M; followed by

(2) Culturing for 2 to 5 days in a medium comprising the BMP inhibitor, a TGF inhibitor and a GSK-3 inhibitor, wherein the TGF inhibitor is present at a concentration of 1 to 5 μ M.

9. The method of claim 1, wherein the suspension culture in step (a) is free of a feeder layer.

10. The method of claim 1, wherein differentiating the neural spheroid into the hnns in step (b) comprises culturing the neural spheroid in suspension culture in a neural medium comprising a GSK-3 inhibitor and a sonic hedgehog pathway agonist, and wherein the method comprises supplementing the neural medium with FGF 4.

11. The method of claim 10, wherein the GSK-3 inhibitor is CHIR 99021.

12. The method of claim 10 or claim 11, wherein the sonic hedgehog (SHH) pathway agonist is a Smooth Agonist (SAG).

13. The method of claim 10, wherein the neural spheroids are cultured in neural media comprising the GSK-3 inhibitor and SHH pathway agonist for 1 to 3 weeks, and wherein the neural media is supplemented with FGF4 after 2 to 10 days.

14. The method of claim 1, wherein step (b) further comprises supplementing the neural medium with at least one of the compounds selected from the group consisting of: brain Derived Neurotrophic Factor (BDNF), NT3, L-ascorbic acid 2-trisodium phosphate (AA), N6, 2' -O-dibutyryladenosine 3',5' -cyclic monophosphate sodium salt (cAMP), cis-4, 7,10,13,16, 19-docosahexaenoic acid (DHA) and DAPT,

optionally, wherein the neural medium is supplemented with the at least one compound after 1 to 3 weeks.

15. The method of claim 1, wherein differentiating the neural spheroid into the hnns in step (b) comprises:

(1) culturing the neural spheroids in suspension culture in a neural medium comprising a GSK-3 inhibitor and a sonic hedgehog pathway agonist for 2 to 10 days;

(2) supplementing the neural medium with FGF 4; and

(3) culturing the neural spheroids in suspension culture in neural medium comprising a GSK-3 inhibitor, a sonic hedgehog pathway agonist and FGF4 for 1 to 3 weeks.

16. The method of claim 15, further comprising the step (3) of:

(4) culturing the neural spheroids in suspension culture in a neural medium comprising FGF4 and at least one of the compounds selected from the group consisting of: brain Derived Neurotrophic Factor (BDNF), NT3, L-ascorbic acid 2-trisodium phosphate (AA), N6, 2' -O-dibutyryladenosine 3',5' -cyclic monophosphate sodium salt (cAMP), cis-4, 7,10,13,16, 19-docosahexaenoic acid (DHA) and DAPT; and

(5) culturing said neurospheres in suspension culture in a neural medium comprising said at least one compound in the absence of FGF4 for at least 1 week.

17. The method of claim 1, wherein the maintaining of the hnns in step (c) is performed in neural medium in the absence of growth factors.

18. The method of claim 1, wherein the hnns is maintained in step (c) for at least 1 week.

19. The method of claim 1, wherein the cells of the hRNS comprise at least one allele associated with a neurological or psychiatric disorder.

20. The method of claim 17, wherein the neurological or psychiatric disorder is selected from the group consisting of: schizophrenia, affective disorder, and Autism Spectrum Disorder (ASD), optionally wherein the affective disorder is major depressive disorder, bipolar disorder, or anxiety disorder.

21. A human metakaryotic spheroid (hRNS) comprising a human serotonergic neuron, wherein said hRNS is capable of remaining in suspension culture for at least 1 month without adhering to a surface.

22. A human midcircuitry spheroid (hnns) obtained by the method of claim 1.

23. A method for producing a cortical-raphanine class assembly (hCS-hRNS) in vitro, the method comprising:

(i) (ii) (a) inducing first human pluripotent stem cells in suspension culture to a neural fate to provide first neurospheres;

(b) differentiating the first neural spheroid into a human midkine spheroid (hRNS),

(ii) (ii) (a) inducing human pluripotent stem cells in a second suspension culture to a neural fate to derive second neural spheroids;

(b) differentiating said second neural spheroid into a cortical spheroid (hCS); and

(iii) culturing the hRNS and hCS in neural culture medium under conditions that allow cell fusion such that the cortical-raphe assembly comprises human serotonergic neurons projecting between the hRNS and hCS, and neurons from the hCS projecting into the hRNS.

24. The method of claim 23, wherein human neurons form bidirectional projections between the hnns and hCS.

25. The method of claim 23, wherein the first and/or second human pluripotent stem cell is an induced human pluripotent stem cell.

26. The method of claim 23, wherein the inducing of the first human pluripotent stem cells into the neural fate in suspension culture in step (a) comprises culturing in a medium comprising a Bone Morphogenetic Protein (BMP) inhibitor and a transforming growth factor beta (TGF β) inhibitor, and wherein the method comprises supplementing the medium with a GSK-3 inhibitor.

27. The method of claim 23, wherein the BMP inhibitor is doxorphine and the TGF inhibitor is SB-431542.

28. The method of claims 23-26, wherein the GSK-3 inhibitor is CHIR 99021.

29. The method of claim 23, wherein the medium comprising the BMP inhibitor and TGF inhibitor is cultured for 2 to 10 days.

30. The method of claim 29, wherein the medium is supplemented with a GSK-3 inhibitor after 1 to 2 days, the medium comprising the BMP inhibitor, TGF inhibitor, and GSK-3 inhibitor, optionally wherein the medium comprising the BMP inhibitor, TGF inhibitor, and GSK-3 inhibitor is cultured for 4 to 8 days.

31. The method of claim 30, wherein culturing in the medium comprising the BMP inhibitor, TGF inhibitor, and GSK-3 inhibitor comprises:

(1) culturing for 2 to 5 days in a medium comprising the BMP inhibitor, a TGF inhibitor, and a GSK-3 inhibitor, wherein the TGF inhibitor is present at a concentration of 5 μ M to 20 μ M; followed by

(2) Culturing for 2 to 5 days in a medium comprising the BMP inhibitor, a TGF inhibitor and a GSK-3 inhibitor, wherein the TGF inhibitor is present at a concentration of 1 to 5 μ M.

32. The method of claim 23, wherein differentiating said first neural spheroid into said hRNS in step (i) (b) comprises culturing said neural spheroid in suspension culture in a neural medium comprising a GSK-3 inhibitor and a SHH pathway agonist, and wherein said method comprises supplementing said neural medium with FGF 4.

33. The method of claim 32, wherein the GSK-3 inhibitor is CHIR 99021.

34. The method of claims 32-33, wherein the SHH pathway agonist is a Smooth Agonist (SAG).

35. The method of claim 32, wherein the neural spheroids are cultured in neural media comprising the GSK-3 inhibitor and sonic hedgehog pathway agonist for 1 to 3 weeks, and wherein the neural media is supplemented with FGF4 after 2 to 10 days.

36. The method of claim 23, wherein step (i) (b) further comprises supplementing the neural medium with at least one of the compounds selected from the group consisting of: brain Derived Neurotrophic Factor (BDNF), NT-3, L-ascorbic acid 2-trisodium phosphate (AA), N6, 2' -O-dibutyryladenosine 3',5' -cyclic monophosphate sodium salt (cAMP), cis-4, 7,10,13,16, 19-docosahexaenoic acid (DHA) and DAPT,

optionally, wherein the neural medium is supplemented with the at least one compound after 1 to 3 weeks.

37. The method of claim 23, wherein differentiating the first neural spheroid into the hRNS in step (i) (b) comprises:

(1) culturing the neural spheroids in suspension culture in a neural medium comprising a GSK-3 inhibitor and a sonic hedgehog pathway agonist for 2 to 10 days;

(2) supplementing the neural medium with FGF 4; and

(3) culturing the neural spheroids in suspension culture in neural medium comprising a GSK-3 inhibitor, a sonic hedgehog pathway agonist and FGF4 for 1 to 3 weeks.

38. The method of claim 37, further comprising step (3) of:

(4) culturing the neural spheroids in suspension culture in a neural medium comprising FGF4 and at least one of the compounds selected from the group consisting of: brain Derived Neurotrophic Factor (BDNF), NT3, L-ascorbic acid 2-trisodium phosphate (AA), N6, 2' -O-dibutyryladenosine 3',5' -cyclic monophosphate sodium salt (cAMP), cis-4, 7,10,13,16, 19-docosahexaenoic acid (DHA) and DAPT; and

(5) culturing said neurospheres in suspension culture in a neural medium comprising said at least one compound in the absence of FGF4 for at least 1 week.

39. The method of claim 23, wherein step (iii) comprises culturing the hnns and hCS under conditions that allow cell fusion for at least 3 days.

40. The method of claim 23, wherein step (iii) comprises culturing the hnns and hCS in direct physical contact with each other.

41. The method of claim 23, wherein the suspension culture in step (i) (a) or step (ii) (a) is a feeder-layer-free culture condition.

42. The method of claim 23, wherein the cells of the cortical-synuclein assembly comprise at least one allele or genetic event associated with a neurological or psychiatric disorder.

43. The method of claim 23, wherein the first or second human pluripotent stem cell comprises at least one allele or genetic event associated with a neurological or psychiatric disorder.

44. The method of claims 42-43, wherein the neurological or psychiatric disorder is selected from the group consisting of: schizophrenia, affective disorder, and Autism Spectrum Disorder (ASD), optionally wherein the affective disorder is major depressive disorder, bipolar disorder, or anxiety disorder.

45. A cortical-centromere assembly (hCS-hRNS) comprising a centromere spheroid (hRNS) fused to a cortical spheroid (hCS), wherein the hCS-hRNS comprises human serotonergic neurons that project between the hRNS and hCS and form a human neuromodulation circuit, and wherein the hCS-hRNS is capable of remaining in suspension culture for at least 4 weeks without adhering to a surface.

46. A cortical-raphe nucleus-like assembly produced by the method of any one of claims 23-44.

47. A method of determining the effect of a candidate agent on serotonergic modulation on cortical neural circuits, the method comprising: contacting the candidate agent with a cortical-raphanine assembly (hCS-hRNS) according to claims 45-46; and determining the effect of the candidate agent on the ability of serotonergic neurons to modulate cortical neural circuit function,

optionally, wherein the cells of hCS-hnns comprise at least one allele or genetic event associated with a neurological or psychiatric disorder, further optionally, wherein the neurological or psychiatric disorder is selected from the group consisting of: schizophrenia, affective disorders, and Autism Spectrum Disorders (ASD).

48. The method of claim 47, wherein the candidate agent is a Selective Serotonin Reuptake Inhibitor (SSRI).

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